powerpoint presentation · 2018-07-22 · pequenas pedras.a bola de cristal é um instrumento das...

10/22/2012

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“Structure isn’t everything, but it sure helps”

Brian W. Matthews

Biophysical Journal (Annual Meeting Abstracts) 2001

10/22/2012 5 V POSLATAM - Buzios 10/22/2012 6 V POSLATAM - Buzios

A. de Saint-Exupéry, 1957, «Le Petit Prince», Gallimard,

Paris

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“Light is a messenger, carrying a story about the form of the object…”

W. L. Bragg,

Mackenzie Davidson Memorial Lecture

November 14, 1928



“ … appreciation of ours present limitation in the area of

protein interactions provides a salutary antidote to the

impression of perfection that the student of biochemistry is

likely to receive from the amount of structural detail of the

proteins that X-ray crystallography, and more recently

magnetic resonance, have made available”

Gregorio Weber

in “Protein Interactions”, 1992

Chapter II - The Chemical Potentials of Proteins


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Cristalomancia Origem: Wikipédia, a enciclopédia livre.

Cristalomancia é o uso dos cristais ou pedras semipreciosas para supostamente prever o futuro; podendo ser por meio da uma bola de cristal ou de jogos com pequenas pedras.A bola de cristal é um instrumento das artes adivinhatórias, muito popular entre os videntes. A cristalomancia é também muito praticada pelas bruxas,

mais com um propósito filosofico.

Prática dessa mancia 1.Antes de tudo, purifique o cristal que será usado. 2.Relaxe, feche os olhos, tire o peso de seus ombros. 3.Abra os olhos, deixe sua mente ver o que tem dentro do bola.

Lista de fatores O significado de cada fator dentro da bola de cristal.

•Nuvens Violetas: harmonia e tranqüilidade •Nuvens Azuis: conquista e felicidade •Nuvens Verde: lucro e prosperidade

•Nuvens Amarelas: duvidas esclarecidas em breve •Nuvens Laranjas: decisões difíceis definitivas •Nuvens Vermelhas: obstáculos e agitação


• Introduction • Crystallization Techniques

• Symmetry • Diffraction

• Structure elucidation


Intr Macr Xt, McPherson 10/22/2012 13 V POSLATAM - Buzios

Abordagem moderna / contemporânea de

descoberta e desenvolvimento de fármacos


RMN Cryo-EM & Single Particle


http://pt.wikipedia.org/wiki/Cristal

http://www.fei.com/TitanETEM/Overview.aspx

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1. Crescer cristais

3. Resolver fase e refinar estrutura

2. Medir difração

Cristalografia e difração de raios-X

10/22/2012 17 V POSLATAM - Buzios •© 2006

•Academic

Press


The first crystal structure of a protein molecule

• 1962: Max Ferdinand Perutz and Sir John Cowdery

Kendrew win the Nobel Prize in Chemistry for their

studies on the structures of globlular proteins.

• The structure of myoglobin was solved by MIR.

(Max Perutz, 1914-2002)


http://en.wikipedia.org/wiki/John_Kendrew

F10

ITC, IUCr

http://it.iucr.org/figures/

John Kendrew with model of myoglobin in progress. Copyright by

the Laboratory of Molecular Biology in Cambridge, England.

The 2 Å-resolution map of sperm-whale myoglobin was

represented by coloured Meccano-set clips on a forest of vertical rods. (Figure provided by M. F. Perutz) 10/22/2012 20 V POSLATAM - Buzios

This is a Kendrew wire model of alcohol dehydrogenase that is

about to undergo a round of rebuilding by Maelle Cambillau.

http://journals.iucr.org/d/issues/2004/12/01/ba5066/index.html 10/22/2012 21 V POSLATAM - Buzios

•© 2006

•Academic

Press


http://almaz.com/nobel/chemistry/chemistry.html


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•© 2006

•Academic

Press




Cristalografia

• Breve introdução

• Objetivos


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• Tutorial interativo (Java App) Bragg: http://www.bmsc.washington.edu/people/merritt/bc530/bragg/

• Tutorial interativo (Java App) Bragg: http://www.bmsc.washington.edu/people/merritt/bc530/bragg/


Cristalização

• Cristalização:

– screeninig com fatoriais;

– otimização;

“Hanging drop” “caixinhas”


Arranjo Cristalino


Cristal

• Estrutra altamente ordenada;

• Alta resolução;

• Precisão da posição dos átomos;


Representação esquemática da fonte de raios X

Ânodo

Rotátório(Cu)

Fonte primária

de raios X

Monocromador

Feixe

focalizado

Detector


Padrão de difração

• raios-X difratados pela densidade eletrônica dos átomos da amostra. 10/22/2012 34 V POSLATAM - Buzios

http://www.bmsc.washington.edu/people/merritt/bc530/bragg/

http://www.bmsc.washington.edu/people/merritt/bc530/bragg/

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Mapa de densidade eletrônica


Mapa de densidade eletrônica


Resolução

• Qualidade do cristal;

• Certeza da localização

de um grupo químico no espaço;

(d)



Cristalização - Métodos


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Four major steps in crystallization

• Obtain large amounts of pure protein

samples

• Choose a protein buffer in which the protein is both soluble and stable

• Bring protein solution to supersaturation

where spontaneous nucleation can take place

• Crystal growth now begins


Soluções para cristalização

kits comerciais

• Hampton

• Jena

• Emerald

• Qiagem

• Sigma

• …




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Tutoriais de cristalização

• Rigaku -

http://www.rigaku.com/protein/crystall ization.html

• Hampton -

http://hamptonresearch.com/experiments.aspx

• Google it !



http://www.rigaku.com/protein/crystallization.html

http://hamptonresearch.com/experiments.aspx

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Robos de cristalização

• Métodos: Sitting / Capilar / Hanging / Batch

• Robo de preparo de solução

• Métodos de pipetagem: spray / toque

(dispensing)









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Solubility As a rule, protein solubility will usually increase as you add salt

to your aqueous solution, then begin to decrease when the salt

concentration gets high enough to compete with the protein for

hydration (interaction with water molecules).

Diagram from the website of Alan Clark, Victoria University of Wellington, New Zealand

http://www2.vuw.ac.nz/staff/alan_clark/teaching/index.htm

HbCO

(carboxy hemoglobin)

solubility as a function

of ionic strength in the

presence of several

different ty pes of salts

10/22/2012 61 V POSLATAM - Buzios Intr Macr Xt, McPherson


Supersaturation

Supersaturation can be achieved by adding more of a substance

(to a solution) than can normally be dissolved. This is a

thermodynamically unstable state, achieved most often in

protein crystallography by vapor diffusion or other slow

evaporation techniques.

Zone 1 - Metastable zone. The solution may not nucleate for a long time

but this zone will sustain growth.

It is frequently necessary to add a seed crystal.

Zone 2 - Nucleation zone. Protein crystals nucleate and grow.

Zone 3 - Precipitation zone. Proteins do not nucleate but precipitate out

of solution.

Diagram from the website for The University of Reading, Course FS460

Investigating Protein Structure and Function 10/22/2012 63 V POSLATAM - Buzios

Diagrama de Fase


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Diagrama de Fase


•Academic

Press



Nucleation

A phenomenon whereby a “ nucleus”, such as a dust particle, a

tiny seed crystal, or commonly in protein crystallography, a

small protein aggregate, starts a crystallization process.

Nucleation poses a large energy barrier, which is easier to

overcome at a higher level of supersaturation.

Common difficulties:

1. If supersaturation is too high, too many nuclei form, hence

an overabundance of tiny crystals.

2. In supersaturated solutions that don’t experience

spontaneous nucleation, crystal growth often only occurs in

the presence of added nuclei or “ seeds”. 10/22/2012 68 V POSLATAM - Buzios

Crystal Growth

Adding single molecules to the

surfaces of the nucleating lattice.

Illustrated here through the work of

Li and Nadarajah of

The Macromolecular

Crystallization Laboratory

at the University of Toledo.

AFM image of individual lysozyme molecules on

the (110) face of a

tetragonal crystal. (Li and Nadarajah)

The growth steps and growth units of Lysozyme. The growth steps are at least bimolecular in height. The minimum

growth unit for this step must be a tetramer corresponding to a single turn of

the 43 helix as shown here.(Nadarajah) H. Li, M.A. Perozzo, J.H. Konnert, A, Nadarajah & M.L. Pusey, Acta Crystallographica, D55,

1023-1035 (1999). 10/22/2012 69 V POSLATAM - Buzios

Cessation of growth

Caused by the development of growth defects or the

approach of the solution to equilibrium.

Mother liquor

The solution in which the crystal exists - this is often

not the same as the original crystallization screening

solution, but is instead the solution that exists after

some degree of vapor diffusion, equilibration through

dialysis, or evaporation. 10/22/2012 70 V POSLATAM - Buzios

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Major factors that affect crystallization

1) Purity of proteins

2) Protein concentration

3) Starting conditions (make-up of the protein solution)

4) Precipitating agent (precipitant)

5) Temperature

6) pH

7) Additives: Detergents, reducing agents, substrates, co-factors,

etc.



ITC, F 10/22/2012 73 V POSLATAM - Buzios

1) Purity of proteins

Sources of heterogeneity (other than unrelated

proteins and nucleic acids as contaminants):

• Partial proteolysis products

• Oxidation of cysteines

• Deamidation of Asn and Gln to Asp and Glu

• Post-translational modifications

• Oligomerization

• Isoforms

• Misfolded population

• Structural flexibility


2) Protein concentration

Consistency and reproducibility are the major issues

with protein concentration - you should have a reliable

assay for determining the concentration.

• Extinction coefficient for tryptophan

• Bradford Assay (BSA is used as a standard)

E. coli expression systems are crystallographers’ most

commonly used method of obtaining protein. Problems can

arise from low expression yields:

• Cytotoxic - your protein is killing your E. coli

• Unstable plasmid or mRNA

• Protein is misfolded (coexpress with GroEL?)

• Some common eukaryotic codons are rare in E. coli 10/22/2012 75 V POSLATAM - Buzios

3) Starting conditions (make-up of

the protein solution)

The main point is to KNOW what your starting conditions

are for purposes of reproducibility.


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4) Precipitating agent (precipitant)

Salts

Ammonium sulfate

Sodium chloride

Potassium phosphate

Organic reagents

MPD

Isopropanol

Polyethylene glycol

PEG 4000

PEG 6000

PEG 8000




5) Temperature

Temperature affects protein stability and also the dynamics of how

protein solution reaching supersaturated states.

Ideally:

• An individual cry stal screen should be kept at constant temperature

• Each set of conditions should be screened at several temperatures

• The easiest are 4 C and room temperature, also try 12 or 15 C


6) pH

Surface charges affect “ crystal packing”.

(Crystal packing refers to the spatial arrangement of

molecules within the crystal, particularly in

reference to their relationships to one another.)

Hydrophobic interactions are less important than

electrostatic interactions in crystal packing.



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7) Additives:

Sometimes you can increase the stability of your protein,

and/or the homogeneity of its conformation by having

relevant additives present in the crystal screen:

• Detergents

• Reducing agents

• Substrates

• Co-factors

• etc.


Still no crystals after thorough

screening. Now what?

• New constructs

Deletion mutants

Complexes with substrates

Protein complex with Fab fragments

Homologous proteins

Fab


Common Methods for Crystallization:

Vapor Diffusion

Slow Evaporation

Dialysis

ITC, F 10/22/2012 86 V POSLATAM - Buzios


Comparative Studies of Protein

Crystallization by Vapour-Diffusion and

Microbatch Techniques

Acta Crystallographica Section D

Volume 54 Issue 1, Pages 8 – 15

Naomi E.Chayen

http://www3.interscience.wiley.com/cgi

-bin/fulltext/119126302/PDFSTART


http://www3.interscience.wiley.com/cgi-bin/fulltext/119126302/PDFSTART



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Variáveis

• pH

• Temperatura

• Tampào / precipitantes / ligantes

• Pressão

• Método de cristalização

• Construção de proteína

• Variáveis combinadas



• Tradicional

• Dos mais usados

VD: hanging


•Academic

Press


Hanging Drop Vapor Diffusion

Most popular method among

protein cry stallographers. 1. Crystal screen buffer is the

well solution (0.5 - 1 mL) 2. Drop (on siliconized glass cover slip) is 1/2 protein

solution, 1/2 cry stal screen buffer (6-10 L). So, the concentration of precipitant in the drop is 1/2 the

concentration in the well. 3. Cover slip is inverted over the top of the well and sealed

with vacuum grease (airtight).

4. The precipitant concentration in the drop will equilibrate with the

precipitant concentration in the well via vapor diffusion.



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VD: hanging

A Standard crystallization support

B The surface of the crystallization support will easily

accommodate 4 drops.

C The screw-in crystallization supports allow easy setup and

reopening.

http://www.qiagen.com/ 10/22/2012 95 V POSLATAM - Buzios

VD: hanging

http://www.qiagen.com/

CrystalSupport X-Seal


VD: hanging

A Hanging drop in standard crystallization support -

side view and top view

B: Hanging drop in Dropguard crystallization

support

Flattened Drops for Easier Visualization Support for Multidrop Experiments

http://www.qiagen.com/ 10/22/2012 97 V POSLATAM - Buzios

Diagrama esquemático demonstrando o

aparato utilizado para cristalização de

proteínas pelo método da gota pendente.

(arte: Ronaldo Nagem)


VD: sitting

• Placas

• Interface

• Coleta cristal


•Academic

Press


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Sitting Drop Vapor Diffusion

Same basic principles

as in hanging drop

method, except the drop

containing your sample

sits on a bridge within

the well. This allows for

a larger sample size (20 -

40 L), however protein

is frequently precious to

the crystallographer, so

there isn’t that much

demand for a larger sample

size.




VD: sitting

www.douglas.co.uk 10/22/2012 104 V POSLATAM - Buzios

VD: sitting


VD: sitting


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VD: sitting


VD: sitting




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Microbatch


• - Start with very pure protein

• - Create a supersaturated the solution

• - Wait… mins , days, weeks, …

reservoir volume ~ 1 mL droplet volume ~ 2L

Crescendo cristais….


Videos


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Interpreting the Results of the Crystallization Experiment


Cristal !


• É proteína ou sal ? Proteína !

• Não é sal !..... Difrata ?

• Difrata !......... É mosaico / twinning ?

• Boa mosaicidade ! Resiste à coleta ?

• Coletado ! Como resolver fase ?

– Subst molec: que modelo usar ?

– Mét. direto: Se-Met ? Metais ? Difrata ?....

Cristal !


© 2006

Academic Press

Difração

- Organização do cristal

- Tamanho (massa)

- Feixe de luz (fluxo de fontos)

Mosaicidade

- “multiplos cristais”

- quanto menor, melhor (< 1o)


Resolução

• Qualidade do cristal;

• Certeza da localização de um grupo químico no espaço;

(d)


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Resolução em experimento de difração de cristais de

macromoléculas e aplicabilidade

• Novos enovelametos / estruturas: < 3.5 Å

• Interação com macromoléculas: < 3.5 Å

• Efeitos conformaci onais de estruturas conhecidas: < 3.0 Å

• Interação com pequenas moléculas (fármacos, etc): < 2.5 Å

• Duplas ocupânci as, dinâmica, detalhes de interação intramolecul ar: <

2.0 Å

• Refinamento de parâmetros estruturais e validação de metodol ogia: <

1.5 Å

10/22/2012 125 V POSLATAM - Buzios http://www.hamptonresearch.com/stuff/gallery.html 10/22/2012 126 V POSLATAM - Buzios


Properties of protein crystals

• Soft, easy to crush

• Contain large solvent channels

– Relatively large organic and inorganic molecules

can diffuse inside

• Anisotropic physical properties

– Birefrigence due to anisotropic refraction indices

• Ability to diffract X-ray due to regular spaced lattices


Varian (Oxford Diffraction)

Quem é proteína, quem é sal ?! (corante: azul de metileno, Izit ™ p/ Hampton)


Padrão de difração

• raios-X difratados pela densidade eletrônica dos átomos da amostra.


http://www.hamptonresearch.com/stuff/gallery.html

http://upload.wikimedia.org/wikipedia/commons/a/aa/Methylene_blue.png

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http://www.oxford-diffraction.com/pdf/PXScanner_handout.pdf 10/22/2012 131 V POSLATAM - Buzios

http://www.oxford-diffraction.com/pdf/PXScanner_handout.pdf 10/22/2012 132 V POSLATAM - Buzios

Varian (Oxford Diffraction)


The oscillation equipment Rotates the crystal about an axis () perpendicular to the

x-ray beam (and normal to the goniometer). The diffraction

pattern from a crystal is a 3-D pattern, and the crystal must

be rotated in order to observe all the diffraction spots.

This nice diagram also comes from Bernhard Rupp’s Crystallography

101 website: http://www-structure.llnl.gov/Xr ay/101index.ht ml 10/22/2012 134 V POSLATAM - Buzios

Crystal Mounting

Capillary tubes

(Glass or Quartz)

Cryo-loops

(thin nylon)


Coleta – montagem de cristal

• Capilares

• Loop

• Pás

• Coleta direto do aparato


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•© 2006

•Academic

Press


Capilar

http://it.iucr.org/figures/Fafig5o1o2o2.gif 10/22/2012 138 V POSLATAM - Buzios

Princ Ptn x-ray diffr,

Drenth, 3rd Ed 10/22/2012 139 V POSLATAM - Buzios 10/22/2012 140 V POSLATAM - Buzios



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Preparo de cristais para coleta

• Soaking com tampão-mãe – retirar tp ptn

• Soaking com ligante

• Soaking com crioprotetor / óleo

• Quebra

• Remoção de ‘pele’ / microcristais


10/22/2012 147 V POSLATAM - Buzios F10 ITC, IUCr 10/22/2012 148 V POSLATAM - Buzios

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Coleta a RT:

MiTeGen MicroRT™ Room

Temperature Mounting System


Criocristalografia

• Desenvolvido em

• Capilar

• Loop em capilar



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Água / gelo

Hkl indeces, Bragg spacings d and relative intensities I/Io of reflections observed in powder

diffraction from crystals of hexagonal ice at 98 K as reported by Dowell and Rinfret

(1960)

Note that the relative intensities of the ice rings found in diffraction photographs form

macromolecular crystals often deviate substantially from the values given in the table

hkl D (Å) I/Io

100 3.897 100

002 3.667 75

101 3.441 53

102 2.671 17

110 2.249 39

103 2.072 30

200 1.948 4

112 1.918 18

201 1.883 3

202 1.721 2




F10

ITC, IUCr

http://it.iucr.org/figures/ 10/22/2012 160 V POSLATAM - Buzios


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Recommended pathways for optimizing cryoprotectant

conditions and flash cooling

F10

ITC, IUCr


F10

ITC, IUCr


F10

ITC, IUCr


F10

ITC, IUCr


F10

ITC, IUCr

http://it.iucr.org/figures/ 10/22/2012 165 V POSLATAM - Buzios 10/22/2012 166 V POSLATAM - Buzios






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Sistemas criogênicos

Soprador de N2g

• a partir de N2L

• compressão de N2g do ar


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Crio vs RT

• Vantagem: não usa aditivo crioprotetor,

não ‘congela’ cristal, dispensa acessório criogênico

• Desvantagem: baixa difração, dano por

radiólise


Otimização de cristalização

• Refinamento das condições: elementos precipitantes

(tãmponante, pH, aditivos), temperatura, método

• Otimizar a pureza do material: preparação proteica,

reagentes

• Trocar o suporte (plaquinha cristalográfica - efeito de

superfície)

• Seeding

• Ligantes: estabilizantes da proteína e/ou interação

interproteica (rede cristalográfica)



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Coleta – fontes de raios-X

• Anodo

• Tubo selado

• Sincrotron

• Fita adesiva (http://www.youtube.com/watch?v=LQBjR

F9mX1Y)

• Coleta remota / in situ


INMETRO

Diffraction directly from

cry stallization screening plates

Images from Agilent webs ite

Cu Mo

Single cry stal diffraction Dual wavelength


LNLS

anodo rotatório 10/22/2012 188 V POSLATAM - Buzios

… o rder a

di f fractometer


http://www.youtube.com/watch?v=LQBjRF9mX1Y

http://www.youtube.com/watch?v=LQBjRF9mX1Y

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Geradores: Funciomanento

• Tubo Selado

• Anodo rotatório

• Sincrotron


•Academic

Press




Drenth, 3rd Ed 10/22/2012 200 V POSLATAM - Buzios

ITC, F 10/22/2012 201 V POSLATAM - Buzios Princ Ptn x-ray diffr,

Drenth, 3rd Ed 10/22/2012 202 V POSLATAM - Buzios

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Drenth, 3rd Ed 10/22/2012 203 V POSLATAM - Buzios Intr Macr Xt, McPherson


•© 2006

•Academic

Press


Rigaku

• Anodo rotatório

• Tubo selado

• Ambos, opções Cu, Mo

• Detector RaxisIV (Rigaku) ou Mar – IP / CCD



Bruker - AXS

• Tubo selado

• Detectores: CCD

• Fontes: Cu / Mo


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Bruker - AXS


Agilent (Oxford Diffraction)

• Tubo selado

• Detectores: CCD

• Fontes: Cu / Mo







The LNLS 1.37 GeV electron storage in December 7, 1996 [3]. All twelve dipolar magnets are visible. Inside the ring two klystrons and their associated modulators are apparent; they feed the LINAC located underground with RF

http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0103-97331997000400002

Braz. J. Phys. vol.27 n.4 São Paulo Dec. 1997 10/22/2012 214 V POSLATAM - Buzios




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Brazilian Synchrotron Light Laboratory (LNLS)

Campinas - Brazil

10/22/2012 215 V POSLATAM - Buzios ITC, F 10/22/2012 216 V POSLATAM - Buzios

Typical time dependence of electron current in the LNLS storage ring (April 1997).



17 Fev 2004 (ainda um pouco oscilante…)


Photon flux from the bending magnets of the storage ring and from the planned 7 Tesla wavelength-shifter.


Braz. J. Phys. vol.27 n.4 São Paulo Dec. 1997 10/22/2012 219 V POSLATAM - Buzios http://www.scielo.br/scielo.php?pid=S1516-14392002000100002&script=sci_arttext

Mat. Res. vol.5 no.1 São Carlos Jan./Mar. 2002 10/22/2012 220 V POSLATAM - Buzios







http://www.scielo.br/scielo.php?pid=S1516-14392002000100002&script=sci_arttext



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Picture of the SAXS beamline(3). The beamline passes across the shielding at the left. The sequence of optical components is: mirror chamber (for vertical focusing), first four-slit set, monochromator chamber (for monochromatization and horizontal focusing), second four-slit set, guard slit set, sample

holder, beamstopper and vertical X-ray position-sensitive detector.



Location of the nine beamlines to be opened to users in 1997 (seven) and 1998 (two).



L. A. Bernardes © 10/22/2012 223 V POSLATAM - Buzios

LNLS: general considerations

Linac: 120 MeV (e - injection energy )

Booster: 500 MeV (maximum e - operation energy )

Storage Ring : 1.37 GeV (e - operation energy )

Open facility supported by the Brazilian Science

and Technology Ministery (MCT).

Beginning of operation: July 1997.

Link: http:// www.lnls.br

12 Bending magnet (BM) beamlines open for users:

9 in X-ray range, 3 in UV and soft X-ray range. 3 BM beamlines under commissioning. 1 Wiggler beamline under commissioning for

anomalous diffraction protein crystallography. 2 New insertion device beamlines under construction: ondulator beamline for UV experiments and wiggler

beamline for material science (X-ray experiments). D02A-SAXS2


SAXS – SAS1

• Primeira linha do LNLS

• Agora em outra saída do anel

• Novo detector – 2D

• Mesmo desenho da MX1

• Detalhes da linha antiga:








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SAXS – SAS1


Mat. Res. vol.5 no.1 São Carlos Jan./Mar. 2002 10/22/2012 227 V POSLATAM - Buzios

L. A. Bernardes ©


L. A. Bernardes ©


L. A. Bernardes ©


L. A. Bernardes ©


Detectores: Funcionamento

• Placa de imagem

• CCD





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Rigaku Ultra18X (IFSC, LNLS)

Image Plate Mar345dtb


IP - Mar345dtb


IP - Mar345dtb


IP – R-AXIS V


Mardtb


MX1 – MarCCD


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•© 2006

•Academic

Press


The SX Series are the only

CCD X-ray detectors that are ideal for both synchrotrons and rotating anode X-ray sources. The SX-165 features a

round, 165 mm diameter active area, and a versatile, high-resolution CCD chip.

The CCD chip in the SX-165

is cooled to -70°C and protected inside a sealed vacuum chamber. The resulting dark current, less than 0.01e-/pixel/sec., allows exposures long enough for data

collection from any crystal on any X-ray source. The refrigeration system requires only a standard electrical outlet

and no cooling water.

http://www.mar-usa.com/support/downloads/sx_series.pdf

MX1 – MarCCD


http://www.marresearch.com/marccd.htm 10/22/2012 241 V POSLATAM - Buzios

MX2 - MarMosaic


MX2 - MarMosaic

http://www.marresearch.com/products.mx-series.html 10/22/2012 243 V POSLATAM - Buzios

Quantum





http://www.marresearch.com/products.mx-series.html



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